Prenylquinones are a large group of compounds with lipid affinities comprising, inter alia, plastoquinones, tocopherols and tocotrienols. In plants, prenylquinones are synthesized via the homogentisate pathway.
The most well-known prenylquinone is vitamin E, or α-tocopherol, an essential element of the human or animal diet, in particular that of mammals which do not produce it naturally but have a dietary need thereof. The most recognized effect of vitamin E is its antioxidant action on cell membrane lipids (Epstein et al., 1966, Radical Research 28: 322-335; Kamel-Eldin and Appelqvist, 1996, Lipids 31: 671-701).
Other than vitamin E, it has been demonstrated that tocotrienols, although they are not essential in the human and animal diet, have particularly advantageous antioxidant properties that are more pronounced than those of vitamin E (Kamat et al., 1997, Mol. Cell. Biochem. 170, 131-137). These compounds are in particular known to protect cells against free radicals, and also to prevent the appearance of cardiovascular diseases or of cancers (Packer et al., 2001, J. Nutr. 131(2): 3698-3738). In addition, tocotrienols exhibit anticancer activity by inhibition of estrogen receptor proliferation, an activity that tocopherols do not possess (Guthrie et al., 1997, J. Nutr. 127: 544-548). They also exhibit a much better hypocholesterolemic activity than tocopherols (Pearce et al., 1992, J. Med. Chem. 35: 3595-3606; Qureshi et al., 2001, J. Nutr. 131: 2606-2618), which makes them more capable of combating arteriosclerosis.
Plastoquinones have no known role in human or animal health, but play an essential role in plants. These molecules are present in chloroplast membranes and their function is that of electron transport during the photosynthesis reaction (Grumbach, 1984, Structure Function and Metabolism of plant lipids, Siegenthaler and Eichenberger eds.).
In addition, an increase in the amount of prenylquinones should confer on plants better resistance to oxidative stresses, in particular cold, drought or strong light.
In plants and photosynthetic organisms in general, homogentisate constitutes the aromatic precursor of prenylquinones. Homogentisate is the product of the p -hydroxyphenylpyruvate dioxygenase enzyme (hereinafter referred to as HPPD). In most organisms, HPPDs are enzymes involved in the catabolic degradation pathway of the aromatic amino acid tyrosine (Goodwin, 1972, in Tyrosine Metabolism: The biochemical, physiological and clinical significance of ρ-hydroxyphenylpyruvate oxygenase, Goodwin B. L., ed., Oxford University press, 1-94). HPPDs catalyze the reaction of conversion of para-hydroxyphenylpyruvate (HPP), a tyrosine degradation product, to homogentisate.
Most plants synthesize tyrosine via arrogenate (Abou-Zeid et al. 1995 Applied Env Microb 41: 1298-1302; Bonner et al., 1995 Plant Cells Physiol. 36, 1013-1022; Byng et al., 1981 Phytochemistry 6: 1289-1292; Connely and Conn 1986 Z. Naturforsch 41c: 69-78; Gaines et al., 1982 Plants 156: 233-240). In these plants, the HPP is derived only from the degradation of tyrosine. On the other hand, in organisms such as the yeast Sacharomyces cerevisiae or the bacterium Escherichia coli, HPP is a tyrosine precursor, and it is synthesized by the action of an enzyme, prephenate dehydrogenase (hereinafter referred to as PDH), which converts prephenate to HPP (Lingens et al., 1967 European J. Biochem 1: 363-374; Sampathkumar and Morrisson 1982 Bioch Biophys Acta 701: 204-211). In these organisms, the production of HPP is therefore directly connected to the aromatic amino acid biosynthetic pathway (shikimate pathway), and not to the tyrosine degradation pathway (see Figure 1).
Up until now, three main strategies using genetic engineering have been employed in order to make plants tolerant to herbicides. The first consists in detoxifying the herbicide by transforming the plant with a gene encoding a detoxifying enzyme. This enzyme converts the herbicide, or its active metabolite, into nontoxic degradation products, for instance the enzymes for tolerance to bromoxynil or to baste (EP 242 236, EP 337 899).
The second strategy consists in transforming the plant with a gene encoding the target enzyme that has been mutated in such a way that it is less sensitive to the herbicide, or its active metabolite, for instance the glyphosate tolerance enzymes (EP 293 356; Padgette et al., 1991, J. Biol. Chem. 266: 33).
The third strategy consists in overexpressing the sensitive target enzyme in such a way as to produce, in the plant, large amounts of target enzyme, if possible much greater than the amount of herbicide entering the plant. This strategy makes it possible to maintain a sufficient level of functional enzyme, despite the presence of its inhibitor. This third strategy has been implemented and has made it possible to obtain plants tolerant to HPPD inhibitors (WO 96/38567). In addition, this simple strategy of overexpression of the sensitive (non-mutated) target enzyme was used successfully for the first time for conferring on plants tolerance at an agronomic level to a herbicide.
It is also known that most HPPD-inhibiting herbicides are competitive inhibitors with respect to the substrate, that bind slowly and virtually irreversibly (Ellis et al., 1996, Chem. Res. Toxicol. 9: 24-27; Viviani et al., 1998, Pestic. Biochem. Physiol. 62: 125-134). Their mode of action therefore consists in competing with the HPP by binding preferentially to its binding site. The result of this binding is an arrest of homogentisate synthesis by the cell.